Fibrilliform-free insulin composition

ABSTRACT

Compositions and methods are disclosed for treating a patient with insulin, wherein a composition in a form suitable for nasal delivery to a patient comprises a therapeutically effective amount of insulin, a permeation enhancer, a liquid carrier, and an acid present in said composition in an amount sufficient to provide an acidic pH, but not greater than a pH of 4.5, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid. Preferably the composition is essentially-free of citrate(s) and essentially-free of phosphate(s). The composition being thus designed with the intent to provide a composition that is essentially-free of fibrilliform (or beta-sheet) insulin polymorphs.

This application claims priority to U.S. Provisional Application Ser. No. 60/811,913, filed Jun. 8, 2006, the disclosures of which are hereby incorporated by reference in their entireties.

This invention relates to compositions and methods for the delivery of insulin to patients, and more particularly to the nasal delivery of insulin to patients.

Insulin is generally used to treat patients that suffer from diabetes.

Insulin is a globular, oligomeric protein containing two chains, A (21 residues) and B (30 residues). A globular protein is one wherein the polypeptide chains are tightly folded into compact spherical or globular shapes. Proteins having two or more polypeptide chains are commonly referred to as oligomeric proteins; and their component chains are commonly referred to as subunits or protomers. Oligomeric proteins usually contain an even number of polypeptide chains that may be identical or different in their length or amino acid sequencing.

Most traditionally, insulin is delivered to patients by way of injection.

U.S. Pat. No. 7,112,561 discloses a nasal composition that contains a combination of insulin and a permeation enhancer, that is maintained at an acidic pH.

However, at an acidic pH fibrillation occurs, which is the tendency of these active ingredients to undergo polymerization through the formation of dimers (“dimerization”) and then to aggregate into crystalline structures that are commonly referred to as fibrils. These “fibrils” are also referred to as fibrilliform (or beta-sheet) insulin polymorphs.

Insulin fibrillation is believed to be promoted by factors that destabilize the classical self-association pathway of monomer to dimer to tetramer to hexamer to higher-order native assemblies, presumably by making the fibrillation-susceptible insulin monomer more available. (See Hua, Qing-xin; Weiss, Michael A.; Mechanism of Insulin Fibrillation: The Structure of Insulin Under Amyloidogenic Conditions Resembles a Protein-Folding Intermediate, J. Biol. Chem., Vol. 279, Issue 20, 21449-21460, May 14, 2004).

In its native form insulin forms a small α-helix-rich globular domain. During the aggregation process there is a transition from the native α-helical-rich secondary structure to a β-sheet-rich structure within the insulin amyloid fibril. The process of insulin fibril formation is known to involve several stages: monomerization of the protein, formation of a partially folded intermediate, nucleation (involving the association of hydrophobic surfaces), and fibril growth. (See Nielsen, Liza; Khurana, Ritu; Coats, Alisa; Frokjaer, Sven; Brange, Jens; Vyas, Sandip; Uversky, Vladimir N.; Fink, Anthony L.; Effect of Environmental Factors on the Kinetics of Insulin Fibril Formation: Elucidation of the Molecular Mechanism; Biochemistry (2001), 40(20), 6036-6046).

It is generally recognized that acidic pH conditions favor the monomer/dimer forms of insulin, while physiological pH conditions favor its tetramer/hexamer forms. (See Nielsen et al.; Probing the Mechanism of Insulin Fibril Formation with Insulin Mutants, Biochemistry, 40 (28), 8397-8409, 2001.) The hexamer oligomer form is believed to prevent insulin from undergoing this fibrillation/misfolding during in vivo storage, and the hexamer is therefore used in clinical formulations. However, acidic conditions generally preclude formation of hexamers (presumably owing to the protonation of a histidine at the trimer interface), and at room temperature insulin retains a native-like conformation at pH 2 and is predominantly dimeric at millimolar concentrations. (See Hua, Qing-xin; Weiss, Michael A.; Mechanism of Insulin Fibrillation: The Structure of Insulin Under Amyloidogenic Conditions Resembles a Protein-Folding Intermediate, J. Biol. Chem., Vol. 279, Issue 20, 21449-21460, May 14, 2004).

It is desirable that the insulin component of a transmembrane delivered formulation should not be in crystalline (aggregated) form when the formulation is administered to the patient, and it is equally desirable that the insulin component in such a transmembrane delivered formulation should be resistant to crystallization (aggregation) while the formulation is manufactured, packaged, stored, refrigerated, or shipped. It is further desirable that little or no “beta pleated sheet” form be allowed to form, since such “beta pleated sheet” form is not only crystalline (known commonly as “fibrilliform” insulin) and thus capable of forming occlusive precipitates, but it is also substantially less biologically active.

In accordance with the invention there is provided a pharmaceutical composition in a form suitable for nasal delivery to a patient, which comprises a therapeutically effective amount of insulin (and/or analogue etc.), a permeation enhancer, and a liquid carrier, wherein the pharmaceutical composition is at an acidic pH, but not greater than a pH of 4.5; wherein the acidic pH of the composition is provided for by the further presence therein of an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid.

In general, the pH of the composition is no greater than 4.5. Preferably the pH of the composition is no greater than 4 nor below 2. The pH is preferably at least 2.

As non-limiting examples of the monovalent inorganic acids that may be used in the invention there may be mentioned hydrochloric acid (as is used in the Example) or hydrobromic acid.

In accordance with the invention there is also provided a pharmaceutical composition in a form suitable for nasal delivery to a patient, which comprises a therapeutically effective amount of insulin (and/or analogue etc.), a permeation enhancer, and a liquid carrier, wherein the pharmaceutical composition is at an acidic pH, but not greater than a pH of 4.5; wherein the acidic pH of the composition is provided for by the further presence therein of an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; and wherein the pharmaceutical composition is essentially-free of citrates.

In accordance with the invention there is further provided a pharmaceutical composition in a form suitable for nasal delivery to a patient, which comprises a therapeutically effective amount of insulin (and/or analogue etc.), a permeation enhancer, and a liquid carrier, wherein the pharmaceutical composition is at an acidic pH, but not greater than a pH of 4.5; wherein the acidic pH of the composition is provided for by the further presence therein of an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; and wherein the pharmaceutical composition is essentially-free of phosphates.

In accordance with the most preferred embodiments of the invention there is provided a pharmaceutical composition in a form suitable for nasal delivery to a patient, which comprises a therapeutically effective amount of insulin (and/or analogue etc.), a permeation enhancer, and a liquid carrier, wherein the pharmaceutical composition is at an acidic pH, but not greater than a pH of 4.5; wherein the acidic pH of the composition is provided for by the further presence therein of an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; and wherein the pharmaceutical composition is essentially-free of citrates and essentially-free of phosphates.

In one embodiment the pharmaceutical compositions are acidified by an acid selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid (preferably acidified by HCl); are essentially-free of citrates; and are essentially-free of phosphates; so as to reduce the formation of the undesirable fibrilliform (or beta-sheet) insulin polymorphs, as compared to compositions wherein acidic conditions are maintained by use of phosphoric acid or citric acid buffers. In a preferred embodiment, the composition is essentially-free of fibrilliform.

In accordance with the invention, there is provided a pharmaceutical composition in a form suitable for nasal delivery to a patient, comprising: a therapeutically effective amount of insulin, a permeation enhancer, and a liquid carrier; wherein the composition has an acidic pH, but not greater than a pH of 4.5; wherein the composition includes an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; said acid being present in an amount sufficient to adjust the pH; wherein the composition is essentially-free of citrate(s); and wherein the composition is essentially-free of phosphate(s).

In a preferred embodiment Applicants have found that the pharmaceutical composition, being acidified by a monovalent inorganic acid(s) (preferably acidified by HCl); being essentially-free of citrates; and being essentially-free of phosphates; reduces the formation of the undesirable fibrilliform (or beta-sheet) insulin polymorphs, as compared to compositions wherein acidic conditions are maintained by use of phosphoric acid or citric acid buffers. In a preferred embodiment, the composition is essentially-free of fibrilliform.

In accordance with the invention, there is provided a method of treating a patient in need of insulin by administering to a patient a pharmaceutical composition in a form suitable for nasal delivery to a patient, comprising: a therapeutically effective amount of insulin, a permeation enhancer, and a liquid carrier; wherein the composition has an acidic pH, but not greater than a pH of 4.5; wherein the composition includes an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; said acid being present in an amount sufficient to adjust the pH.

In accordance with the invention, there is also provided a method of treating a patient in need of insulin by administering to a patient a pharmaceutical composition in a form suitable for nasal delivery to a patient, comprising: a therapeutically effective amount of insulin, a permeation enhancer, and a liquid carrier; wherein the composition has an acidic pH, but not greater than a pH of 4.5; wherein the composition includes an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; said acid being present in an amount sufficient to adjust the pH; and wherein the composition is essentially-free of citrate(s).

In accordance with the invention, there is further provided a method of treating a patient in need of insulin by administering to a patient a pharmaceutical composition in a form suitable for nasal delivery to a patient, comprising: a therapeutically effective amount of insulin, a permeation enhancer, and a liquid carrier; wherein the composition has an acidic pH, but not greater than a pH of 4.5; wherein the composition includes an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; said acid being present in an amount sufficient to adjust the pH; and wherein the composition is essentially-free of phosphate(s).

In accordance with the most preferred embodiments of invention, there is provided a method of treating a patient in need of insulin by administering to a patient a pharmaceutical composition in a form suitable for nasal delivery to a patient, comprising: a therapeutically effective amount of insulin, a permeation enhancer, and a liquid carrier; wherein the composition has an acidic pH, but not greater than a pH of 4.5; wherein the composition includes an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; said acid being present in an amount sufficient to adjust the pH; wherein the composition is essentially-free of citrate(s); and wherein the composition is essentially-free of phosphate(s).

The invention further relates to treating a patient in need of insulin with a combination of insulin, a permeation enhancer, and a liquid carrier, wherein the combination comprises an acid selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; wherein the combination is essentially-free of citrate(s); and wherein the combination is essentially-free of phosphate(s); resulting in the combination being essentially-free of fibrilliform (or beta-sheet) insulin polymorphs; the combination having an acidic pH, but not greater than a pH of 4.5. The combination may be formed ex situ by combining its components (or groups of its components) into a final composition prior to its administration to the patient, or it may be formed in situ by the individual administration of its components (or groups of its components) to the nasal mucosa of the patient.

The pH of the composition is maintained by the use of a suitable acid that does not substantially contribute to the formation of fibrilliform (or beta-sheet) insulin polymorphs, and the composition may be buffered by also adding a suitable base. A base, if used, is preferably a monovalent base, such as sodium hydroxide or potassium hydroxide.

In accordance with another embodiment of the invention, there is provided a pharmaceutical composition for nasal administration to humans, or to warm-blooded animals, comprising: (A) a therapeutically effective amount of insulin, (B) a permeation enhancer, (C) a liquid carrier, and (D) a combination of non-ionic surfactants; wherein the combination of non-ionic surfactants comprises: (i) at least one fatty acid ester of a sugar or sugar alcohol and (ii) at least one pegylated fatty acid ester of a sugar or sugar alcohol; wherein the composition has an acidic pH, but not greater than a pH of 4.5; wherein the acidic pH of the composition is provided by the composition further comprising an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; wherein the composition is essentially-free of citrate(s); and wherein the composition is essentially-free of phosphate(s). The composition being thus designed with the intent to provide a composition that is essentially-free of fibrilliform (or beta-sheet) insulin polymorphs.

In accordance with another embodiment of the invention, there is provided a pharmaceutical composition for nasal administration to humans, or to warm-blooded animals, comprising: (A) a therapeutically effective amount of insulin, (B) a permeation enhancer, and (C) a liquid carrier; wherein the osmolality of the composition is <200 mOsmol/Kg H₂O (preferably the osmolality of the composition is <150 mOsmol/Kg H₂O); wherein the composition has an acidic pH, but not greater than a pH of 4.5; wherein the acidic pH of the composition is provided by the composition further comprising an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; wherein the composition is essentially-free of citrate(s); and wherein the composition is essentially-free of phosphate(s). The composition being thus designed with the intent to provide a composition that is essentially-free of fibrilliform (or beta-sheet) insulin polymorphs.

In accordance with another embodiment of the invention, there is provided a pharmaceutical composition for nasal administration to humans, or to warm-blooded animals, comprising: (A) a therapeutically effective amount of insulin, (B) a permeation enhancer, (C) a liquid carrier, and (D) a combination of non-ionic surfactants; wherein the combination of non-ionic surfactants comprises: (i) at least one fatty acid ester of a sugar or sugar alcohol and (ii) at least one PEGylated fatty acid ester of a sugar or sugar alcohol; and wherein the osmolality of the composition is <200 mOsmol/Kg H₂O (preferably the osmolality of the composition is <150 mOsmol/Kg H₂O); wherein the composition has an acidic pH, but not greater than a pH of 4.5; wherein the acidic pH of the composition is provided by the composition further comprising an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; wherein the composition is essentially-free of citrate(s); and wherein the composition is essentially-free of phosphate(s). The composition being thus designed with the intent to provide a composition that is essentially-free of fibrilliform (or beta-sheet) insulin polymorphs.

While the invention has been thus far described as nasally deliverable compositions and as nasally practiced methods, in alternative embodiments the compositions may be administered intravenously or as part of infusion therapies, and the methods may be practiced intravenously or as part of infusion therapies. In such alternative embodiments the composition is administered in forms hereinabove and hereinbelow described with the exception that the permeation enhancer and surfactants are excluded therefrom. In these embodiments the reduced fibril production will result in less occlusive crystalline insulin occluding the pumps, needles, and tubing upon which IV and infusion therapies rely.

As known in the art “PEG” or “peg” is an abbreviation for polyethylene glycol or polyoxyethylene—polymeric forms of ethylene oxide—either of which may be produced synthetically or derived from animal or vegetable sources. As used hereinabove and hereinbelow, and as understood in the art, the term “pegylated” shall mean that a polyethylene glycol and/or polyoxyethylene chain(s) is covalently attached to a molecule. The pegylated portion of the fatty acid ester of the sugar or sugar alcohol of some of the embodiments of the present invention may include a polymer chain that includes from about 4 to about 25 oxyethylene units.

In one embodiment of the present invention, the pegylated portion of the fatty acid ester of the sugar or sugar alcohol includes a polymer chain that includes about 20 oxyethylene units.

Simple sugars include sucrose, fructose, glucose, galactose, maltose, lactose, and mannose. Fatty acids generally have from 12 to 20 carbon atoms and may be saturated or unsaturated. Common fatty acids include lauric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, and oleic acid. As known in the art, sorbitol, derived from glucose by hydrogenation or electrolytic reduction, is a sugar alcohol. The Span® series of surfactants are esters of sorbitol and fatty acids, and are therefore fatty acid esters of sugar alcohols useful in the non-ionic surfactant combinations of some of the embodiments of the compositions of the instant invention. As known in the art sorbitan is produced through a dehydration process of sorbitol and this intermediate may be esterified with a fatty acid, whereby esters of sorbitan are included within the meaning of the term “fatty acid ester of a sugar or sugar alcohol” as it is used hereinabove and hereinbelow to describe that element of some of the embodiments of the instant invention. Pegylated sorbitan monolaurate (or polyoxyethylene sorbitan monolaurate) sometimes referred to as Polysorbate 20, is included within the meaning of the term “pegylated fatty acid ester of a sugar or sugar alcohol” as the term is used hereinabove and hereinbelow to describe that element of some of the embodiments of the instant invention. In one embodiment, the combination of non-ionic surfactants contains at least one fatty acid ester of a sugar alcohol and at least one pegylated fatty acid ester of a sugar alcohol. In one embodiment the sugar alcohol is sorbitol.

The esterified sugar or sugar alcohol may be esterified with one or more fatty acids, which may be the same or different. In one embodiment, the sugar or sugar alcohol is mono-esterifed.

In some preferred embodiments of the invention, the combination of non-ionic surfactants, comprising (i) at least one fatty acid ester of a sugar or sugar alcohol and (ii) at least one pegylated fatty acid ester of a sugar or sugar alcohol, has a combined hydrophilic-lipophilic balance (HLB) of from about 7 to about 14.

In a particularly preferred embodiment of the invention, the combination of non-ionic surfactants, comprising (i) at least one fatty acid ester of a sugar or sugar alcohol and (ii) at least one pegylated fatty acid ester of a sugar or sugar alcohol, comprises Sorbitan Monolaurate and Polysorbate 20, respectively.

In one embodiment, the hereinabove-described and hereinbelow-described compositions of the invention are hypotonic relative to the red blood cells of the human body. In such an embodiment, in general, the osmolality of the composition is <200 mOsmol/Kg H₂O. Preferably the osmolality of the composition is <150 mOsmol/Kg H₂O. In an embodiment of the invention the osmolality of the composition is <30 mOsmol/Kg H₂O. In another embodiment, the osmolality of the composition is about 25 mOsmol/Kg H₂O.

In another embodiment, the invention further relates to treating a patient in need of insulin with any of the hereinabove described or hereinbelow described pharmaceutical compositions for nasal administration to humans, or to warm-blooded animals, which contain inter alia: insulin, a permeation enhancer, and a liquid carrier; wherein the composition further comprises a combination of non-ionic surfactants; wherein the combination of non-ionic surfactants comprises: (i) at least one fatty acid ester of a sugar or sugar alcohol and (ii) at least one pegylated fatty acid ester of a sugar or sugar alcohol; wherein the composition has an acidic pH, but not greater than a pH of 4.5; wherein the acidic pH of the composition is provided by the composition further comprising an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; wherein the composition is essentially-free of citrate(s); and wherein the composition is essentially-free of phosphate(s). The composition being thus designed with the intent to provide a composition that is essentially-free of fibrilliform (or beta-sheet) insulin polymorphs.

In another embodiment, the invention further relates to treating a patient in need of insulin with any of the hereinabove described or hereinbelow described pharmaceutical compositions for nasal administration to humans, or to warm-blooded animals, which contain inter alia: insulin, a permeation enhancer, and a liquid carrier; wherein the osmolality of the composition is <200 mOsmol/Kg H₂O (preferably the osmolality of the composition is <150 mOsmol/Kg H₂O); wherein the composition has an acidic pH, but not greater than a pH of 4.5; wherein the acidic pH of the composition is provided by the composition further comprising an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; wherein the composition is essentially-free of citrate(s); and wherein the composition is essentially-free of phosphate(s). The composition being thus designed with the intent to provide a composition that is essentially-free of fibrilliform (or beta-sheet) insulin polymorphs. In an embodiment of the invention the osmolality of the composition is <30 mOsmol/Kg H₂O. In another embodiment of the invention the osmolality of the composition is about 25 mOsmol/Kg H₂O.

In another embodiment, the invention further relates to treating a patient in need of insulin with any of the hereinabove described or hereinbelow described pharmaceutical compositions for nasal administration to humans, or to warm-blooded animals, which contain inter alia: insulin, a permeation enhancer, and a liquid carrier; wherein the composition further comprises a combination of non-ionic surfactants; wherein the combination of non-ionic surfactants comprises: (i) at least one fatty acid ester of a sugar or sugar alcohol and (ii) at least one PEGylated fatty acid ester of a sugar or sugar alcohol; and wherein the osmolality of the composition is <200 mOsmol/Kg H₂O (preferably the osmolality of the composition is <150 mOsmol/Kg H₂O); wherein the composition has an acidic pH, but not greater than a pH of 4.5; wherein the acidic pH of the composition is provided by the composition further comprising an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; wherein the composition is essentially-free of citrate(s); and wherein the composition is essentially-free of phosphate(s). The composition being thus designed with the intent to provide a composition that is essentially-free of fibrilliform (or beta-sheet) insulin polymorphs.

While the pharmaceutical composition of the invention has been thus far described above with regard to the use of insulin generally as the active ingredient therein, it is to be understood that it is within the scope of the invention that the term insulin is to be broadly interpreted so as to include all forms of insulin, such as, and without limitation, native insulin, recombinant insulin, proinsulin, and any insulin analogues, derivatives, polymorphs, metabolites, pro-drugs, salts, and/or hydrates.

However, notwithstanding the pharmaceutical composition of the invention having been thus far described above with regard to the use of insulin generally as the active ingredient therein, it is to be further understood that it is also within the scope of the invention that the active ingredient may alternatively be a protein, or any similarly polypeptide-composed molecule, particularly any protein or similarly polypeptide-composed molecule in which fibril formation would render such protein or such similarly polypeptide-composed molecule less biologically-active, or otherwise undesirable, as a component of any life-science related formulation.

Additionally, it is to be further understood that it is also within the scope of the invention that the active ingredient may alternatively be a peptide, or any similarly composed molecule, particularly any peptide or similarly composed molecule in which fibril formation would render such peptide or such similarly composed molecule less biologically-active, or otherwise undesirable, as a component of any life-science related formulation.

Additionally, it is to be further understood that it is also within the scope of the invention that the active ingredient may alternatively be a peptidomimetic, or any similarly composed molecule, particularly any peptidomimetic or similarly composed molecule in which fibril formation would render such peptidomimetic or such similarly composed molecule less biologically-active, or otherwise undesirable, as a component of any life-science related formulation.

Additionally, it is to be further understood that it is also within the scope of the invention that the active ingredient may alternatively be a peptoid, or any similarly composed molecule, particularly any peptoid or similarly composed molecule, in which fibril formation would render such peptoid or such similarly composed molecule less biologically-active, or otherwise undesirable, as a component of any life-science related formulation.

In general, the permeation enhancer that is employed is one that enhances the permeation of the insulin composition through the membrane of a body cavity and in particular through the nasal mucosa.

In a composition containing an effective amount of insulin a preferred permeation enhancer is a compound of the structure:

wherein X and Y are oxygen, sulfur or an imino group of the structure

or ═N—R with the proviso that when Y is the imino group, X is an imino group, and when Y is sulfur, X is sulfur or an imino group, A is a group having the structure

wherein X and Y are defined above, m and n are integers having a value from 1 to 20 and the sum of m+n is not greater than 25, p is an integer having a value of 0 or 1, q is an integer having a value of 0 or 1, r is an integer having a value of 0 or 1, and each of R, R₁, R₂, R₃, R₄, R₅ and R₆ is independently hydrogen or an alkyl group having from 1 to 6 carbon atoms which may be straight chained or branched provided that only one of R₁ to R₆ can be an alkyl group, with the proviso that when p, q and r have a value of 0 and Y is oxygen, m+n is at least 11, and with the further proviso that when X is an imino group, q is equal to 1, Y is oxygen, and p and r are 0, then m+n is at least 11, and said compound will enhance the rate of the passage of the drug across body membranes. Hereinafter these compounds are referred to as enhancers. When R, R₁, R₂, R₃, R₄, R₅ or R₆ is alkyl it may be methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, amyl, hexyl, and the like. Such permeation enhancers are described in U.S. Pat. No. 5,023,252 and U.S. Pat. No. 5,731,303, the disclosures of which are hereby incorporated by reference in their entireties.

Preferably, the enhancer compounds of this invention are the cyclic lactones (the compounds wherein both X and Y are oxygen, (q is 1 and r is 0), the cyclic diesters (the compounds wherein both X and Y are oxygen, and both q and r are 1), and the cyclic ketones (the compounds wherein both q and r are 0 and Y is oxygen). In the cyclic diesters m+n is preferably at least 3. In the cyclic ketones m+n is preferably from 11 to 15 and p is preferably 0.

Enhancers of the above structural formula are referred to herein as “Hsieh enhancers” and are described, for example, in aforementioned U.S. Pat. Nos. 5,023,252 and 5,731,303 (hereinafter “Hsieh Patents”). Such enhancers are lipophilic and are “membrane-compatible,” meaning that they do not cause damage to the membrane on which the composition of the present invention is to be applied (hereinafter “target membrane”). Such enhancers produce also a low level of irritability or no irritability to the target membrane and in fact serve as an emollient.

Preferred enhancers for use in the present invention are macrocyclic enhancers. The term “macrocyclic” is used herein to refer to cyclic compounds having at least 12 carbons in the ring. Examples of preferred macrocyclic enhancers for use in the present invention include: (A) macrocyclic ketones, for example, 3 methylcyclopentadecanone (muscone), 9-cycloheptadecen-1-one (civetone), cyclohexadecanone, and cyclopentadecanone (normuscone); and (B) macrocyclic esters, for example, pentadecalactones such as oxacyclohexadecan-2-one (cyclopentadecanolide, ω-pentadecalactone).

Oxacyclohexadecan-2-one and cyclopentadecanone are especially preferred.

Although the above are preferred permeation enhancers, one of ordinary skill in the art would recognize that the instant teachings would also be applicable to other permeation enhancers. Non-limiting examples of other permeation enhancers useful in the instant invention are the simple long chain esters that are Generally Recognized As Safe (GRAS) in the various pharmacopoeial compendia. These may include simple aliphatic, unsaturated or saturated (but preferably fully saturated) esters, which contain up to medium length chains. Non-limiting examples of such esters include isopropyl myristate, isopropyl palmitate, myristyl myristate, octyl palmitate, and the like. The enhancers are of a type that are suitable for use in a pharmaceutical composition. The artisan of ordinary skill will also appreciate that those materials that are incompatible with or irritating to mucous membranes should be avoided.

The enhancer is present in the composition in a concentration effective to enhance penetration of the insulin, to be delivered, through the membrane. Various considerations should be taken into account in determining the amount of enhancer to use. Such considerations include, for example, the amount of flux (rate of passage through the membrane) achieved and the stability and compatibility of the components in the formulations. The enhancer is generally used in an amount of about 0.01 to about 25 wt. % of the composition, more generally in an amount of about 0.1 to about 15 wt. % of the composition, and in preferred embodiments in an amount of about 0.5 to about 10 wt % of the composition.

The liquid carrier is present in the composition in a concentration effective to serve as a suitable vehicle for the compositions of the present invention. In general, the carrier is used in an amount of about 40 to about 98 wt. % of the composition and in preferred embodiments in an amount of about 50 to about 98 wt. % of the composition.

The insulin compositions of the present invention are preferably delivered as a nasal spray. In such an embodiment, the preferred liquid carrier is water with the insulin being dispersed or dissolved in the water in a therapeutically effective amount.

In one preferred embodiment, the permeation enhancer is emulsified in the aqueous phase that contains the insulin. The emulsification may be effected through the use of one or more suitable surfactants. The selection of a suitable surfactant is deemed to be within the scope of those skilled in the art based on the teachings herein. Essentially any suitable surfactant or mixture of surfactants can be used in the practice of the present invention, including, for example, anionic, cationic, and non-ionic surfactants. Preferred surfactants are non-ionic surfactants, with those having a hydrophilic-lipophilic balance (HLB) of from about 7 to about 14 being particularly preferred. Examples of such non-ionic surfactants are PEG-60 corn glycerides, PEG-20 sorbitan monostearate, phenoxy-poly(ethyleneoxy)ethanol, sorbitan monooleate, and the like. Especially preferred are compendial surfactants such as those described in compendia such as the Food Chemicals Codex, National Formulary, U.S. Pharmacopeia, and the Code of Federal Regulations. It is preferred that the average diameter of the droplets of the emulsion be from about 100 nm to about 20 μm and more preferably from about 400 nm to about 5 μm. In general an individual surfactant is present in an amount no greater than about 5 wt. % of the composition and more generally no greater than about 1.5 wt. % of the composition.

In one preferred embodiment, the emulsified or discontinuous phase that contains the permeation enhancer is in the form of droplets. In general, smaller droplets confer greater stability. Larger droplets may cause instability and decrease shelf-life. In preferred embodiments the droplet size ranges from 0.1 microns to 20 microns and preferably from 0.1 microns to 5 microns.

In general compositions that contain insulin are stored in a refrigerator and such refrigeration may result in crystallization of the permeation enhancer. In order to inhibit or prevent such crystallization, in a preferred embodiment the composition includes one or more crystallization inhibitors to inhibit the crystallization of the permeation enhancer. Crystallization, if allowed to proceed, renders the emulsion unstable and has an adverse effect on shelf life. Preferred crystallization inhibitors function by lowering the temperature at which the involved compound crystallizes. Examples of such crystallization inhibitors include natural oils, oily substances, waxes, esters, and hydrocarbons. Examples of natural oils or oily substances include Vitamin E acetate, octyl palmitate, isopropyl myristate, sesame oil, soybean oil, safflower oil, avocado oil, palm oil, and cottonseed oil. The selection of a suitable crystallization inhibitor is deemed to be within the scope of those skilled in the art from the teachings herein. Preferred crystallization inhibitors function by lowering the temperature at which the permeation enhancer crystallizes.

Inhibitors which are capable of lowering the temperature of crystallization of the involved compound to below about 25° C. are particularly preferred, with those capable of lowering the crystallization of the involved compound to below about 5° C. being especially preferred. Examples of especially preferred crystallization inhibitors for use in inhibiting the crystallization of oxacyclohexadecan-2-one include hexadecane, isopropyl myristate, octyl palmitate, cottonseed oil, safflower oil, and Vitamin E acetate, each of which may be used in pharmaceutical preparations.

The crystallization inhibitor is present in the composition in a concentration effective to inhibit the crystallization of the permeation enhancer. In general the crystallization inhibitor is present in an amount of about 0.001 to about 5 wt. % of the composition, more generally in an amount of from about 0.01 to about 2 wt % of the composition. In one embodiment the crystallization inhibitor is present in an amount of from about 0.1 to about 1 wt. % of the composition. The crystallization inhibitor is one preferably used when the enhancer has a crystallization temperature above about 0 degrees Centigrade. In particular, for example, a crystallization inhibitor is preferably used when the enhancer is pentadecalactone and/or cyclohexadecanone, since these crystallize above room temperature.

The composition of the present invention is generally delivered through a nasal spray applicator. If intra-nasal application is desired, the composition may be placed in an intra-nasal spray-dosing device or atomizer and be applied by spraying it into the nostrils of a patient for delivery to the mucous membrane of the nostrils. A sufficient amount is applied to achieve the desired systemic or localized drug levels. For an intra-nasal spray, up to about 200 microliters is typically applied, with an application of about 50 to about 150 microliters being preferred. One or more nostrils may be dosed and application may occur as often as desired or as often as is necessary. In preferred embodiments, the nasal spray applicator is selected to provide droplets of the composition of a mean size of from about 10 microns to about 200 microns. More generally the droplet size is from about 30 microns to about 100 microns.

The insulin spray composition of the invention is generally employed in a dosing regimen that is dependent on the patient being treated. Thus, the frequency of the use and the amount of the dose may vary from patient to patient. In general, dosing is in an amount (the amount internalized after absorption from the mucosa) of from about 3 IU to about 15 IU and the frequency of dose is 3 to 4 times per day. As known in the art, the treatment of a disease such as diabetes through insulin therapy varies from patient to patient, and based on known insulin therapy and the teachings herein one skilled in the art can select the dosing regimen and dosage for a particular patient or patients.

The composition of the present invention comprises insulin. The insulin is present in the composition in a therapeutically-effective amount. In general the insulin is present in an amount of about 0.1 to about 1.5 wt. % of the composition. In preferred embodiments the insulin is present in an amount of about 0.25 to about 1.25 wt. % of the composition. In more preferred embodiments the insulin is present in an amount of about 0.25 to about 1.0 wt. % of the composition.

Although a preferred embodiment is a preformulated composition, it is also within the scope of the present invention that a patient may be treated with the hereinabove described and/or hereinbelow described combination that is not preformulated; i.e., the insulin in liquid carrier, the enhancer, and an acid, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid; each being essentially-free of citrate(s) and essentially-free of phosphate(s) may be mixed at the time of application, such as where the mixing occurs in an atomizer at the time the composition is sprayed.

It is anticipated that compositions and methods that can diminish protein misfolding and aggregation, such as the inventions hereinabove described and hereinbelow described, could lead to new insulin, peptide, polypeptide, or protein formulations/therapies and/or could improve upon the efficacy of existing insulin, peptide, polypeptide, or protein formulations/therapies, and in the case of insulin itself, may broaden the range of analogs available for use in the treatment of diabetes.

Although, the compositions as hereinabove described and hereinbelow described are designed as hereinabove described and hereinbelow described with the intent to provide compositions that are essentially-free of fibrilliform (or beta-sheet) insulin polymorphs, it is nonetheless within the scope of the hereinabove described and hereinbelow described invention that the formulations may additionally contain fibrillation inhibitors. As non-limiting examples of fibrillation inhibitors that may be used in the making, and/or practicing, of the hereinabove described and hereinbelow described formulations and methodologies there may be mentioned: 1-anilinonaphthalene-8-sulfonic acid (ANS); trimethylamine N-oxide dihydrate (TMAO), 1 M; sucrose, 10%; calcium or zinc ions; polyhydric alcohols; trehalose; betaine; ectoine; and citrulline.

In accordance with an embodiment of the invention, the following composition is prepared:

EXAMPLE 1

Content per 100 μl Component % w/w Dose in mg Recombinant 1 1.0 Human Insulin Pentadecalactone 2 2.0 Cottonseed oil 1 1.0 Sorbitan 0.6 0.6 Monolaurate (Span ® 20) Polysorbate 20 0.7 0.7 (Tween ® 20) Hydrochloric As Needed to acid Achieve a pH of about 3.5 Sodium As Needed to Hydroxide Achieve a pH of about 3.5 Sterile Water for q.s. Injection 

1. An insulin composition in a form suitable for nasal delivery to a patient, comprising: a therapeutically effective amount of insulin, a permeation enhancer, a liquid carrier, and an acid present in said composition in an amount sufficient to provide an acidic pH, but not greater than a pH of 4.5, said acid being selected from the group consisting of: monovalent inorganic acid(s), sulfuric acid, and acetic acid.
 2. The composition of claim 1, wherein said acid is a monovalent inorganic acid.
 3. The composition of claim 2, wherein said composition is essentially-free of citrate(s).
 4. The composition of claim 2, wherein said composition is essentially-free of phosphate(s).
 5. The composition of claim 2, wherein said composition is essentially-free of citrate(s), and wherein said composition is essentially-free of phosphate(s).
 6. The composition of claim 1, wherein said acidic pH is from about 2 to about
 4. 7. The composition of claim 1, wherein said acidic pH is about 3.5.
 8. The composition of claim 1, further comprising a crystallization inhibitor.
 9. The composition of claim 1, wherein said composition is in the form of an emulsion.
 10. The composition of claim 1, wherein said composition is in the form of a spray emulsion.
 11. The composition of claim 1, wherein said permeation enhancer is a macrocyclic permeation enhancer.
 12. The composition of claim 11, wherein said macrocyclic permeation enhancer is selected from the group consisting of macrocyclic ketones and macrocyclic esters.
 13. The composition of claim 12, wherein said macrocyclic ketone is selected from the group consisting of 3 methylcyclopentadecanone (muscone), 9-cycloheptadecen-1-one (civetone), cyclohexadecanone, and cyclopentadecanone (normuscone).
 14. The composition of claim 12, wherein said macrocyclic ester is a pentadecalactone.
 15. The composition of claim 14, wherein said pentadecalactone is oxacyclohexadecan-2-one (cyclopentadecanolide, ω-pentadecalactone).
 16. A product including the pharmaceutical composition of claim 1, wherein said pharmaceutical composition is contained in, and deliverable from, an applicator selected from the group consisting of: nasal spray applicators, intra-nasal spray-dosing devices, and atomizers.
 17. The composition of claim 1, wherein said permeation enhancer is emulsified by a surfactant(s).
 18. The composition of claim 17, wherein said surfactant(s) is/are selected from the group consisting of: non-ionic surfactants and combinations thereof.
 19. The composition of claim 18, wherein said combination of non-ionic surfactants comprises: (i) at least one fatty acid ester of a sugar or sugar alcohol and (ii) at least one pegylated fatty acid ester of a sugar or sugar alcohol.
 20. The composition of claim 19, wherein said combination of non-ionic surfactants, comprising (i) at least one fatty acid ester of a sugar or sugar alcohol and (ii) at least one pegylated fatty acid ester of a sugar or sugar alcohol, has a combined hydrophilic-lipophilic balance (HLB) of from about 7 to about
 14. 21. The composition of claim 1, wherein the osmolality of the composition is <200 mOsmol/Kg H₂O.
 22. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 1. 23. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 2. 24. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 3. 25. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 4. 26. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 5. 27. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 6. 28. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 7. 29. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 8. 30. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 9. 31. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 10. 32. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 11. 33. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 12. 34. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 13. 35. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 14. 36. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 15. 37. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 16. 38. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 17. 39. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 18. 40. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 19. 41. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 20. 42. A method for treating a patient in need of insulin comprising treating said patient by nasally administering to said patient the composition of claim
 21. 